Described herein are methods for identifying and preparing high-affinity nucleic acid ligands and nucleic acid ligands capable of photocrosslinking to target molecules specifically disclosed as nucleic acid ligands to basic Fibroblast Growth Factor155 (bFGF(155)). The method utilized herein for identifying such ligands is called PhotoSELEX, an acronym for Photochemical Systematic Evolution of Ligands by EXponential enrichment. This invention includes high-affinity DNA ligands capable of photocrosslinking bFGF(155). Specifically disclosed are two modified ssDNA ligands to bFGF(155) which exhibited high sensitivity for bFGF(155) comparable to that of commercially available ELISA monoclonal antibodies with an absolute sensitivity of at least 0.058 ppt bFGF(155) under prevailing test conditions. Additionally, the ligands were able to distinguish bFGF(155) from consanguine proteins, Vascular Endothelial Growth Factor (VEGF) and Platelet Derived Growth Factor (PDGF) and from other proteins in serum. Further included within the scope of this invention is a method for determining the exact position of the photocrosslink between the nucleic acid ligand and the target molecule. The present invention includes the use of nucleic acid ligands capable of photocrosslinking to targets as diagnostic reagents.
Effective diagnostics capable of early and accurate detection of marker proteins and other analyte molecules is an area of research emphasis in the pharmaceutical industry. Despite the intensity of recent efforts, many diagnostic protocols still rely on immunochemical detection techniques first described in the early 1970""s (Engvall and Perlman (1971) Immunochem. 8:871-874; Engvall and Perlman (1972) J. Immunol. 109:129-135; Hoffman (1973) J. Allergy Clin. Immunol. 5:303-307; Ljungstrom et al. (1974) Parasitology 69:xxiv). The major technique to evolve from these original investigations is the enzyme-linked immunosorbent assay (ELISA) with the sandwich immunoassay protocol representing the state of the art for large molecule (e.g. protein) detection.
While the sandwich ELISA has been a reliable mainstay for protein and antigen detection, it is often a costly, time consuming and labor intensive technique. Despite efforts to expand and automate the ELISA assay, no system is currently available for the detection of a wide array of important marker proteins in a patient from a single, small sample of the patient""s serum or plasma. In the present research, an in vitro selection methodology called Photochemical Systematic Evolution of Ligands by Exponential Enrichment (photoSELEX) has been employed to identify oligodeoxynucleotide ligands which may supplant antibodies evolved through immunological techniques as diagnostic agents.
Selex
A method for the in vitro evolution of nucleic acid molecules with high affinity binding to target molecules has been developed. This method, Systematic Evolution of Ligands by EXponential enrichment, termed SELEX, is described in U.S. patent application Ser. No. 071536,428, filed Jun. 11, 1990, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment,xe2x80x9d now abandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled xe2x80x9cNucleic Acid Ligands,xe2x80x9d now U.S. Pat. No. 5,475,096 and U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled xe2x80x9cMethods for Identifying Nucleic Acid Ligands,xe2x80x9d now U.S. Pat. No. 5,270,163 (see also WO91/19813), each of which is herein specifically incorporated by reference. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describe a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.
Since its conception more than ten years ago, Systematic Evolution of Ligands by Exponential Enrichment (SELEX) has proven to be a valuable combinatorial technique. The SELEX methodology has been used to successfully identify natural, as well as, modified RNA and ssDNA ligands capable of binding with high affinity and specificity to an impressive array of target molecules (Ellington and Szostak (1990) Nature 346:818-822; Gold et al. (1995) Annu. Rev. Biochem. 64:763-799; Morris et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:2902-2907; Osborne and Ellington (1997) Chem. Rev. 97:349-370; Ruckman et al. (1998) J. Biol. Chem. 273:20556-20567; Schneider et al. (1995) Biochemistry 34:9599-9610; Tuerk and Gold (1990) Science 249:505-510)).
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection theme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield high affinity nucleic acid ligands to the target molecule.
The basic SELEX method may be modified to achieve specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, entitled xe2x80x9cMethod for Selecting Nucleic Acids on the Basis of Structure,xe2x80x9d now abandoned (see U.S. Pat. No. 5,707,796), describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA (See U.S. Pat. No. 5,707,796). U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled xe2x80x9cPhotoselection of Nucleic Acid Ligands,xe2x80x9d now abandoned (see U.S. Pat. No. 5,763,177), describes a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled xe2x80x9cHigh-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine,xe2x80x9d now abandoned (see U.S. Pat. No. 55,580,737), describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed xe2x80x9cCounter-SELEX.xe2x80x9d U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Solution SELEX,xe2x80x9d now abandoned, (see U.S. Pat. No. 5,567,588) and U.S. patent application Ser. No. 08/792,075, filed Jan. 31, 1997, entitled xe2x80x9cFlow Cell SELEX,xe2x80x9d now U.S. Pat. No. 5,861,254, describe SELEX-based methods which achieve highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. U.S. patent application Ser. No. 07/964,624, filed Oct. 21, 1992, entitled xe2x80x9cNucleic Acid Ligands to HIV-RT and HIV-1 Rev,xe2x80x9d now U.S. Pat. No. 5,496,938, describes methods for obtaining improved nucleic acid ligands after the SELEX process has been performed. U.S. patent application Ser. No. 08/400,440, filed Mar. 8, 1995, entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Chemi-SELEX,xe2x80x9d now U.S. Pat. No. 5,705,337, describes methods for covalently linking a ligand to its target.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or delivery. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. Specific SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled xe2x80x9cHigh Affinity Nucleic Acid Ligands Containing Modified Nucleotides,xe2x80x9d now abandoned (see U.S. Pat. No. 5,660,985), that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2xe2x80x2-positions of pyrimidines, as well as specific RNA ligands to thrombin containing 2xe2x80x2-amino modifications. U.S. patent application Ser. No. 08/134,028, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2xe2x80x2-amino (2xe2x80x2-NH2), 2xe2x80x2-fluoro (2xe2x80x2-F), and/or 2xe2x80x2-O-methyl (2xe2x80x2-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled xe2x80x9cNovel Method of Preparation of Known and Novel 2xe2x80x2 Modified Nucleosides by Intramolecular Nucleophilic Displacement,xe2x80x9d now abandoned, describes oligonucleotides containing various 2xe2x80x2-modified pyrimidines. PCT/US98/00589, filed Jan. 7, 1998, entitled xe2x80x9cBioconjugation of Oligonucleotides,xe2x80x9d (WO98/30720), describes a method for identifying bioconjugates to a target comprising nucleic acid ligands derivatized with a molecular entity exclusively at the 5xe2x80x2-position of the nucleic acid ligands.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. patent application Ser. No. 08/284,063, filed Aug. 2, 1994, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX,xe2x80x9d now U.S. Pat. No. 5,637,459 and U.S. patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: Blended SELEX,xe2x80x9d respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules. The full text of the above described patent applications, including but not limited to, all definitions and descriptions of the SELEX process, are specifically incorporated herein by reference in their entirety.
In the PhotoSELEX variation of SELEX, a modified nucleotide activated by absorption of light is incorporated in place of a native base in either RNA- or in ssDNA-randomized oligonucleotide libraries. See U.S. patent application Ser. No. 09/459,553, filed Dec. 13, 1999; U.S. Pat. Nos. 6,001,577; and 5,763,177, each entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: Photoselection of Nucleic Ligands and Solution SELEX;xe2x80x9d and U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled xe2x80x9cPhotoselection of Nucleic Acid Ligands,xe2x80x9d now abandoned, each of which is specifically incorporated herein by reference in its entirety). One such modified nucleotide whose photochemistry is particularly well-suited for this purpose is 5-bromo-2xe2x80x2-deoxyuridine (BrdU) (Meisenheimer and Koch (1997) Crit. Rev. Biochem. Mol. Biol. 32:101-140). The 5-bromouracil chromophore absorbs ultraviolet (UV) light in the 310 nm range where native chromophores of nucleic acids and proteins do not absorb or absorb very weakly. The resulting excited singlet state intersystem crosses to the lowest triplet state which specifically crosslinks with aromatic and sulfur-bearing amino acid residues of a protein target in suitable proximity (Dietz and Koch (1987) Photochem. Photobiol. 46:971-8; Dietz and Koch (1989) Photochem. Photobiol. 49:121-9; Dietz et al. (1987) J. Am. Chem. Soc. 109:1793-1797; Ito et al. (1980) J. Am. Chem. Soc. 102:7535-7541; Swanson et al. (1981) J. Am. Chem. Soc. 103:1274-1276). Crosslinking may also occur via excitation of an aromatic residue of the protein in proximity to the bromouracil chromophore (Norris et al (1997) Photochem. Photobiol. 65:201-207). Of particular importance, excited bromouracil in DNA is relatively unreactive in the absence of a proximal, oriented, reactive amino acid (Gott et al. (1991) Biochemistry 30:6290-6295; Willis et al. (1994) Nucleic Acids Res. 22:4947-4952; Norris et al. (1997) Photochem. Photobiol. 65:201-207) or nucleotide residue (Sugiyama et al. (1990) J. Am. Chem. Soc. 112:6720-6721; Cook and Greenberg (1996) J. Am. Chem. Soc. 118:10025-10030). The importance of orientation is evident in crystal structures of protein-nucleic acid complexes which show a lock and key arrangement of the bromouracil chromophore with the aromatic amino acid residue to which it crosslinks (Horvath et al. (1998) Cell 95:963-974; Meisenheimer and Koch (1997) Crit. Rev. Biochem. Mol. Biol. 32:101-140).
Basic fibroblast growth factor (bFGF(155)) is a 155-amino acid member of the Fibroblast Growth Factor family of polypeptides, having the following primary amino acid sequence (SEQ ID NO:99):
bFGF(155) is a multifunctional effector for many cells of mesenchymal and neuroectodermal origin (Rifkin and Moscatelli (1989) J. Cell Biol. 109:1; Baird and Bohlen (1991) in Peptide Growth Factors and Their Receptors (Spom, M. B. and Roberts, A. B., eds.); pp. 369-418, Springer, N.Y.; Basilico and Moscatelli (1992) Adv. Cancer Res. 59:115). It is one of the most studied and best characterized members of a family of related proteins that also includes acidic FGF (Jaye et al. (1986) Science 233:541; Abraham et al. (1986) Science 233:545), int-2 (Moore et al. (1986) EMBO J. 5:919), kFGF/hst/KS3 (Delli-Bovi et al. (1987) Cell 50:729; Taira et al (1987) Proc. Natl. Acad. Sci. USA 84:2980), FGF-5 (Zhan et al. (1988) Mol. Cell. Biol. 8:3487), FGF-6 (Marics et al. (1989) Oncogene 4:335) and keratinocyte growth factor/FGF-7 (Finch et aL (1989) Science 245:752).
In vitro, bFGF stimulates cell proliferation, migration and induction of plasminogen activator and collagenase activities (Presta et al. (1986) Mol. Cell. Biol. 6:4060; Moscatelli et al. (1986) Proc. Natl. Acad. Sci. USA 83:2091; Mignatti etal. (1989) J. Cell Biol. 108:671). In vivo, it is one of the most potent inducers of neovascularization. Its angiogenic activity in vivo suggests a role in tissue remodeling and wound healing, but also in some disease states that are characterized by pathological neovascularization, such as tumor proliferation, tumor metastasis, diabetic retinopathy and rheumatoid arthritis (Folkman and Klagsbrun (1987) Science 235:442; Gospodarowitz (1991) Cell Biology Reviews 25:307).
Although bFGF does not have a signal sequence for secretion, it is found on both sides of the plasma membrane, presumably being exported via exocytosis (Vlodavsky et al. (1991) Trends Biol. Sci. 16:268; Mignatti and Rifkin (1991) J. Cell. Biochem. 47:201). In the extracellular matrix, it is typically associated with a fraction that contains heparan sulfate proteoglycans. Indeed, heparin affinity chromatography has been a useful method for purification of this and other heparin-binding growth factors. In cell culture, bFGF binds to low- and high-affinity sites. The low-affinity sites are composed of cell-associated heparan sulfate proteoglycans to which bFGF binds with approximately nanomolar affinity (Moscatelli (1987) J. Cell. Physiol. 131:123). All biological effects of bFGF are mediated through interaction with the high-affinity binding sites (10-100 pM) that represent the dimeric tyrosine kinase FGF receptor (IJeno et aL (1992) J. Biol. Chem. 267:1470). Five FGF receptor genes have been identified to date, each of which can produce several structural variants as a result of alternative mRNA splicing (Armstrong et al. (1992) Cancer Res. 52:2004; Ueno et al. (1992) J. Biol. Chem. 267:1470). There is by now substantial evidence that the low- and the high-affinity binding sites act cooperatively in determining the overall affinity of bFGF. Experiments with mutant cell lines that are deficient in glycosaminoglycan synthesis (Yayon et al. (1991) Cell 64:841) or heparitinase treated cells (Rapraeger et al. (1991) Science 252:1705) have shown that binding of either cell-associated heparan sulfate or, in its absence, exogenously added heparin to bFGF is required for signaling via the tyrosine kinase receptor. Recent resolution of observed Kd into its kinetic components demonstrates that while the association rates of bFGF to the low- and the high-affinity sites are comparable, the dissociation rate of bFGF from the cell surface receptor is 23-fold slower than that for the cell-associated heparan sulfate (Nugent and Edelman (1992) Biochemistry 31:8876). The slower off-rate, however, is only observed when the receptor is bound to the cell surface suggesting that simultaneous binding to both sites contributes to the overall high-affinity binding. This is plausible in light of the observation that the heparin-binding and the receptor-binding sites are located on adjacent, but separate regions of the molecule, as determined from the recently solved X-ray crystal structure of bFGF (Zhang et al. (1991) Proc. Natl. Acad. Sci. USA 88:3446; Eriksson et al. (1991) Proc. Natl. Acad. Sci. USA 88:3441; Ago et al. (1991) J. Biochem. 110:360, Zhu et al. (1991) Science 251:90).
The idea that bFGF antagonists may have useful medicinal applications is not new (reviewed in Gospodarowicz (1991) Cell Biology Reviews 25:307). bFGF is now known to play a key role in the development of smooth-muscle cell lesions following vascular injury (Reidy et al. Circulation, Suppl. III 86:III43). Overexpression of bFGF (and other members of the FGF family) is correlated with many malignant disorders (Halaban et al. (1991) Ann. N. Y. Acad. Sci. 638:232; Takahashi et al. (1990) Proc. Natl. Acad. Sci. USA 87:5710; Fujimoto et al. (1991) Biochem. Biophys. Res. Commun. 180:386) and recently, neutralizing anti-bFGF antibodies have been found to suppress solid tumor growth in vivo by inhibiting tumor-linked angiogenesis (Hori et al. (1991) Cancer Res. 51:6180). Notable in this regard is the recent therapeutic examination of suramin, a polysulfated naphthalene derivative with known antiprotozoal activity, as an anti-tumor agent. Suramin is believed to inhibit the activity of bFGF through binding in the polyanion binding site and disrupting interaction of the growth factor with its receptor (Middaugh et al. (1992) Biochemistry 31:9016; Eriksson et al. (1991) Proc. Natl. Acad. Sci. USA 88:3441). In addition to having a number of undesirable side effects and substantial toxicity, suramin is known to interact with several other heparin-binding growth factors which makes linking of its beneficial therapeutic effects to specific drug-protein interactions difficult (La Rocca et al. (1990) Cancer Cells 2:106). Anti-angiogenic properties of certain heparin preparations have also been observed (Folkman et al. (1983) Science 221:719; Crum et al. (1985) Science 250:1375) and these effects are probably based at least in part on their ability to interfere with bFGF signaling. While the specific heparin fraction that contributes to bFGF binding is now partially elucidated (Ishai-Michaeli et al. (1992) Biochemistry 31:2080; Turnbull et aL (1992) J. Biol. Chem. 267:10337), a typical heparin preparation is heterogeneous with respect to size, degree of sulfation and iduronic acid content. Additionally, heparin also affects many enzymes and growth factors.
Described herein are the uses of photoaptamers in a diagnostic system. Photoaptamers can be evolved to numerous targets and can be used to covalently bind the target to a surface for direct detection. Use of a photoaptamer as a capture molecule in a diagnostic assay adds an extra dimension of specificity and supplants the need for sandwich assays. The photoaptamer is attached to any suitable solid support as described in U.S. patent application Ser. No. 09/561,465, filed Jun. 12, 2000 and U.S. patent application Ser. No. 08/990,436, filed Jun. 15, 1997, both entitled xe2x80x9cNucleic Acid Ligand Diagnostic Biochip,xe2x80x9d which are specifically incorporated herein by reference in their entirety. A sample containing unknown substances (e.g. proteins) can be applied to the solid support containing immobilized photoaptamers and an affinity association is formed if the sample contains the target for the photoaptamer. After appropriate incubation, and an optional mild wash the solid support is irradiated with UV light causing covalent bond formation between the photoaptamer and the target. Very aggressive washing can than take place (e.g. with strong detergents for denaturants) to eliminate non-specific binding. Finally, the amount of bound target can be directly determined. If the target is a protein, the detection can be made using a universal protein stain capable of conjugation to the amino acids of the covalently bound protein. Other suitable detection methods, such as SPR, are also compatible with the use of photoaptamers.
Also described herein are methods for identifying and preparing high-affinity nucleic acid ligands and nucleic acid ligands capable of photo crosslinking to target molecules specifically disclosed as nucleic acid ligands to basic Fibroblast Growth Factor155 (bFGF(155)). The present invention includes methods of identifying and producing nucleic acid ligands to human basic Fibroblast Growth Factor155 (bFGF(155)) and the nucleic acid ligands so identified and produced. In particular, DNA sequences are provided that are capable of photocrosslinking bFGF(155). Specifically included in the invention are the DNA ligand sequences shown in Tables 1 and 2 and FIG. 2A (SEQ ID NOS:4-98). The photocrosslinking nucleic acid ligands form tight Michaelis complexes with the targets. The photocrosslinking step enhances the specificity of the protein-nucleic acid ligand interaction.
In one variation, the method of the present invention comprises preparing a candidate mixture of nucleic acid sequences which contain photoreactive groups; contacting the candidate mixture with a target molecule wherein nucleic acid sequences having increased affinity to the target molecule bind the target molecule, forming nucleic acid-target molecule complexes; irradiating the nucleic acid-target molecule mixture, wherein some nucleic acids incorporated in nucleic acid-target molecule complexes crosslink to the target molecule via the photoreactive functional groups; taking advantage of the covalent bond to partition the crosslinked nucleic acid-target molecule complexes from free nucleic acids in the candidate mixture; and identifying the nucleic acid sequences that were photocrosslinked to the target molecule. The process can further include the iterative step of amplifying the nucleic acids that photocrosslinked to the target molecule to yield a mixture of nucleic acids enriched in sequences that are able to photocrosslink to the target molecule.
More specifically, the present invention includes the DNA ligands to bFGF(155) identified according to the above-described method, including those ligands shown in Tables 1 and 2 and FIG. 2A (SEQ ID NOS:4-98). Also included are nucleic acid ligands to bFGF(155) that are substantially homologous to any of the given ligands and that have substantially the same ability to bind bFGF(155). Further included in this invention are nucleic acid ligands to bFGF(155) that have substantially the same structural form as the ligands presented herein and that have substantially the same ability to bind bFGF(155).
In another variation of this embodiment of the present invention, nucleic acid sequences containing photoreactive groups are selected through SELEX for a number of rounds in the absence of irradiation, resulting in a candidate mixture with a partially enhanced affinity for the target molecule. PhotoSELEX is then conducted with irradiation to select ligands able to photocrosslink to the target molecule.
The present invention also includes an improved method for determining the position of the photocrosslink between the nucleic ligand and the target molecule. In this variation of the present invention the photocrosslinked amino acid is identified by sequencing the photo-crosslinked amino acid nucleic acid ligand complex by Mass Spectrometry and the photo-crosslinked base is identified by Maxam-Gilbert sequencing.
The present disclosure provides non-limiting examples which are illustrative and exemplary of the invention. Other partitioning schemes and methods of selecting nucleic acid ligands through binding and photocrosslinking to a target molecule will be become apparent to one skilled in the art from the present disclosure.